CONVERSION OF THE SUBSTRATE-SPECIFICITY OF MOUSE PROTEINASE GRANZYME-B

Mouse granzyme B is the prototypic member of a subfamily of serine pro teinases expressed in cytolytic lymphocytes. Molecular modelling of gr anzyme B indicated that the side chain of Arg 208 partially fills the specificity pocket, thus predicting the preference of this enzyme for substrates containing acidic side chains, a feature unique among eukar yotic serine proteinases. Replacement of Arg 208 with glycine results in an enzyme lacking this activity, but which is able to hydrolyze hyd rophobic substrates. These results demonstrate unequivocally that the substrate preference of granzyme B is determined by a positive charge in the specificity pocket and also represent one of the few examples o f rational and efficient alteration of serine proteinase substrate-spe cificity following a single amino acid substitution.